Hepatitis C virus (HCV) has emerged as a major cause of human liver disease, with ~3% of the world population persistently infected with and more than one million new cases of infection reported ...annually. In most cases, HCV escapes the immune system and establishes a chronic infection. In the long term, these chronic carriers are at high risk of developing life-threatening liver disease, including cirrhosis and hepatocellular carcinoma. Primary human hepatocytes isolated from patient biopsies represent the most physiologically relevant cell culture model for hepatitis C virus (HCV) infection, but these primary cells are not readily accessible, display individual variability, and are largely refractory to genetic manipulation. Hepatocyte-like cells differentiated from pluripotent stem cells provide an attractive alternative as they not only overcome these shortcomings but can also provide an unlimited source of noncancerous cells for both research and cell therapy. Despite its promise, the permissiveness to HCV infection of differentiated human hepatocyte-like cells (DHHs) has not been explored. We therefore developed a novel infection model based on DHHs derived from human embryonic (hESCs) and induced pluripotent stem cells (iPSCs). DHHs generated in chemically defined media under feeder-free conditions were subjected to infection by both HCV derived in cell culture (HCVcc) and patient-derived virus (HCVser). Pluripotent stem cells and definitive endoderm were not permissive for HCV infection whereas hepatic progenitor cells were persistently infected and secreted infectious particles into culture medium. RNA interference directed toward essential cellular cofactors, such as CyPA and PI4K, in stem cells resulted in HCV-resistant hepatocyte-like cells after differentiation. Interestingly, we also identified a defined transition during the hepatic differentiation process when the cells become permissive for HCV infection. Permissiveness to infection was correlated with induction of the liver-specific microRNA-122 and modulation of cellular factors that affect HCV replication. Further studies using microarray analysis of gene expression profile between non-permissive and permissive cells revealed activation of other putative proviral factors and downregulation of antiviral factors. We then focused on CIDEB, a liver specific gene, whose expression was upregulated during the transition stage. Knocking-down CIDEB by shRNA in hESCs had little effect on the differentiation of the modified cells toward functional hepatocytes, but could inhibit the infection of DHHs by HCVcc. Similar inhibition of HCV infection was also observed when CIDEB was knocked down in Huh-7.5 cells, by the same shRNA and a commercial siRNA. Subsequent detailed studies showed that CIDEB is not required for the steps including viral particle attachment, HCVpp entry, RNA translation and replication, virion assembly and secretion, but involved in the fusion step when viral envelope proteins fuse with endosomal membrane and release the viral RNA into cytosol. CIDEB was also found to be required for infection of Huh-7.5 cells by dengue virus (DENV), through a similar mechanism by facilitating membrane fusion. Surprisingly, upon HCV and DENV particles entry, early endosome markers (Rab5 and EEA1) could be induced to re-distribute to the surface of lipid droplets, colocalizating with CIDEB, further supporting the importance of CIDEB during membrane fusion process. HCV, but not DENV infection could downregulate CIDEB expression in infected cells, through a posttranscriptional manner. Finally, knockout of CIDEB also effectively protected Huh-7.5 cells from being infected by both HCV and DENV. Taken together, the ability to infect cultured cells directly with HCVcc and HCV patient serum, to study defined stages of viral permissiveness, and to produce genetically modified cells with desired phenotypes all have broad significance for host pathogen interactions and cell therapy. Meanwhile, our study also identified a liver-specific HCV entry cofactor that facilitates membrane fusion with a new mechanism and contributes to HCV's hepatic tropism. CIDEB and its interaction with HCV may serve as targets for future anti-HCV therapy.
Interferon regulatory factor 7 (IRF7) is a potent transcription factor of type I interferons (IFNs) and IFN stimulated genes (ISGs) and is known as the master regulator of type I IFN-dependent immune ...responses. Because excessive responses could harm the host, IRF7 itself is delicately regulated at the transcriptional, translational, and posttranslational levels. Modification of IRF7 by small ubiquitin-related modifiers (SUMOs) has been shown to regulate IFN expression and antiviral responses negatively, but the specific E3 ligase needed for IRF7 SUMOylation has remained unknown. As reported here, we have identified the tripartite motif–containing (TRIM) protein 28 (TRIM28) as a binding partner of IRF7. We have demonstrated that TRIM28 also interacts with the SUMO E2 enzyme and increases SUMOylation of IRF7 both in vivo and in vitro, suggesting it acts as a SUMO E3 ligase of IRF7. Unlike the common SUMO E3 ligase, protein inhibitor of activated STAT 1(PIAS1), the E3 activity of TRIM28 is specific to IRF7, because it has little effect on IRF7’s close relative IRF3. TRIM28 is therefore, so far as we know, the first IRF7-specific SUMO E3 reported. TRIM28-mediated SUMOylation of IRF7 is increased during viral infection, and SUMOylation of transcription factors usually results in transcriptional repression. Overexpression of TRIM28 therefore inhibits IRF7 transactivation activity, whereas knockdown of TRIM28 has the opposite effect and potentiates IFN production and antiviral responses. Collectively, our results suggest that TRIM28 is a specific SUMO E3 ligase and negative regulator of IRF7.
In this thesis, stochastic volatility models with Lévy processes are treated in parameter calibration by the Carr-Madan fast Fourier transform (FFT) method and pricing through the partial ...integro-differential equation (PIDE) approach. First, different models where the underlying log stock price or volatility driven by either a Brownian motion or a Lévy process are examined on Standard & Poor's (S&P) 500 data. The absolute percentage errors show that the calibration errors are different between the models. Furthermore, a new method to estimate the standard errors, which can be seen as a generalization of the traditional error estimation method, is proposed and the results show that in all the parameters of a stochastic volatility model, some parameters are well-identified while the others are not. Next, the previous approach to parameter calibration is modified by making the volatility constrained under the volatility process of the model and by making the other model parameters fixed. Parameters are calibrated over five consecutive days on S&P 500 or foreign exchange (FX) options data. The results show that the absolute percentage errors do not get much larger and are still in an acceptable threshold. Moreover, the parameter calibrating procedure is stabilized due to the constraint made on the volatility process. In other words, it is more likely that the same calibrated parameters are obtained from different initial guesses. Last, for the PIDEs with two or three space dimensions, which arise in stochastic volatility models or in stochastic skew models, it is in general inefficient or infeasible to apply the same numerical technique to different parts of the system. An operator splitting method is proposed to break down the complicated problem into a diffusion part and a jump part. The two parts are treated with a finite difference and a finite element method, respectively. For the PIDEs in 1-D, 2-D and 3-D cases, the numerical approach by the operator splitting is carried out in a reasonable time. The results show that the operator splitting method is numerically stable and has the monotonicity preserving property with fairly good accuracy, when the boundary conditions at volatility are estimated by Neumann conditions.
Optimisation based shape from shading （SFS） is sensitive to initialization： errors in initialization are a signifi- cant cause of poor overall shape reconstruction. In this paper, we present a method ...to help overcome this problem by means of user interaction. There are two key elements in our method. Firstly, we extend SFS to consider a set of initializations, rather than to use a single one. Secondly, we efficiently explore this initialization space using a heuristic search method, tabu search, guided by user evaluation of the reconstruction quality. Reconstruction results on both synthetic and real images demonstrate the effectiveness of our method in providing more desirable shape reconstructions.
Thesis (Ph. D.) -- University of Maryland, College Park, 2005.
Thesis research directed by: Applied Mathematics and Scientific Computation Program. Title from t.p. of PDF. Includes bibliographical ...references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
Noble metal nanoparticles, such as gold or silver nanoparticles and nanorods, exhibit unique photonic, electronic and catalytic properties. Functionalization of noble metal nanoparticles with ...biomolecules (e.g., protein and DNA) produces systems that possess numerous applications in catalysis, delivery, therapy, imaging, sensing, constructing nanostructures and controlling the structure of biomolecules. In this paper, the recent development of noble metal nanoparticle-biomolecule conjugates is reviewed from the following three aspects: (1) synthesis of noble metal nanoparticle-biomolecule systems by electrostatic adsorption, direct chemisorption of thiol derivatives, covalent binding through bifunctional linkers and specific affinity interactions; (2) the photonic properties and bioactivation of noble metal nanoparticle-biomolecule conjugates; and (3) the optical applications of such systems in biosensors, and medical imaging, diagnosis, and therapy. The conjugation of Au and Ag nanoparticles with biomolecules and the most recent optical applications of the resulting systems have been focused on.
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